The applications cited supra disclose, inter alia, the characterization and preparation of various hormone and hormone-like receptors, including the glucocorticoid, mineralocorticoid, thyroid hormone, and retinoic acid receptors, and analogs of such receptors. These receptors are members of the steroid/thyroid hormone superfamily of receptors. This superfamily is now known, from numerous publications in the art which have described details of such receptors and DNAs that encode them, to include also, inter alia, the estrogen receptor, the progesterone receptor, and the vitamin D3 receptor.
In PCT International Application No. US89/05419 (corresponding to U.S. patent application Ser. No. 278,614, filed on 30 November 1988) are disclosed hormone or hormone-like receptor analogs, for example, analogs of steroid receptors, thyroid hormone receptors, and retinoic acid receptors, including those of the human species, where advantage is provided by enhancement, over that of the corresponding naturally occurring receptor, of the trans-acting, transcription-activation or transcription-enhancement activity. Such enhancement is provided by changes in domains, of the naturally occurring receptors, that were found to be necessary for the transcription-activation or transcription-enhancement activities of the receptors.
It is known, for example, that the glucocorticoid receptor belongs to the large, steroid/thyroid hormone superfamily of ligand-dependent, transcription factors that have diverse roles in homeostasis, reproduction, development, growth, function of the immune system and function of the central and peripheral nervous systems, among others. Comparison of complementary DNAs encoding receptors of this superfamily, as well as mutational analyses of these DNAs, have identified certain functional domains within the receptor molecules responsible respectively for DNA binding, hormone binding and nuclear localization. See Evans, et al., Science 240, 889 (1988) for a review of this subject matter. In the case of the glucocorticoid receptor, the so-called DNA binding domain spans some sixty-six amino acids and is highly conserved among various species and this domain has been found to be required in order to activate transcription. See Hollenberg, et al., Cell 49, 39 (1987), Miesfeld, et al., Science 236, 423 (1987), Danielsen, et al., Mol.Endo 1, 816 (1987), Kumar, et al., Cell 51, 941 (1987), Gronemeyer, EMBO J. 6, 3985 (1987), and Waterman, et al., Mol.Endo 2, 14 (1988). This region has been found to contain nine invariant cysteine residues and, although the contribution of each cysteine residue to overall function as well as the actual structure of this domain, remain unknown, it has been proposed that these cysteine residues coordinate two zinc ions to form two DNA-binding, so-called "zinc-finger," domains which result in a tertiary structure thought responsible for localization and binding of the glucocorticoid receptor to the requisite DNA site. Similar zinc-finger structures are present in other receptors of the steroid/thyroid hormone receptor superfamily. See Klug, et al., Tr.Biochem.Sci 12, 464 (1987), Bens, et al., Cell 52, 1 (1988), and Evans, supra.
In a location nearer the carboxyl-terminal end of the receptor molecule, distal from the DNA binding region of the molecule, is the so-called ligand-binding domain. In the absence of ligand (e.g., hormone or hormone-analog which complexes with the receptor at the ligand-binding domain), the ligand-binding domain functions to block transcription-affecting activity of the receptor. Thus, presence of the requisite hormone relieves the inhibition of the receptor to such activity. Deletion of the ligand-binding domain from a receptor of the steroid/thyroid hormone superfamily has been found to produce a hormone-independent transcription activator. See Godowski, et al., Nature 325, 365 (1987), Hollenberg, et al., supra, Kumar, et al., supra, Danielsen et al., supra, and Adler et al., Cell 52, 685 (1988).
In contrast to these two domains, the domain of a receptor lying towards the amino-terminal region from the DNA binding domain is poorly understood both as to structure and function. This domain nearer the amino-terminus is extremely variable both in size and in composition among the various receptors. See Evans, supra. The domain may contribute to the heterogeneity of receptor function, despite the overall similarity otherwise of the receptors of the superfamily. See Kumar et al., supra, and Tora et al., 333, 185 (1988).
Despite extensive analysis, some of which has been reported in the scientific literature, the region(s) that are responsible (in conjunction with the DNA binding of the receptor) for the transcription activation or enhancement caused by a receptor remains poorly characterized. Transcription-activation (or transcription-enhancement) domains can be defined as regions of a receptor molecule that, when combined with the DNA binding functional domain, increase productive transcription initiation by RNA polymerases at a promoter affected by the receptor. See Sigler, Nature 333, 210 (1988), Brent et al., Cell 43, 729 (1985), Hope et al., Cell 46, 885 (1986), Ma et al., Cell 48, 847 (1987), Ma et al., Cell 51, 113 (1987), Lech et al., Cell 52, 179 (1988), and Hope et al., Nature 333, 635 (1988).
Previous research on the human glucocorticoid receptor by linker scanning mutagenesis identified two regions outside of the DNA binding region having a role in transcription activation. These regions were defined as tau.sub.1 and tau.sub.2. Giguere et al., Cell 46, 645 (1986). Further research from these laboratories has also resulted in the report of a co-localization of transcription-activation and DNA-binding functions. See Hollenberg et al., supra, Miesfeld, et al., supra, Danielsen et al., Supra, and Waterman et al., supra. As a composite, this research has given rise merely to a picture, for a receptor of the steroid/thyroid hormone superfamily, that is becoming increasingly modular, with discrete domains, each contributing to the functions of ligand-binding, DNA-binding and "trans-activation" (by which is meant, herein, trans-acting transcription-activation (or enhancement). However, the picture based upon extant literature does not adequately portray the dynamic nature of the activities of the steroid/thyroid hormone receptors and how the various domains participate in the cascade of events initiated by ligand-binding and consummated by promoter-specific trans-activation.
Further, although previous research has identified functional "domains", there has been little systematic effort to identify amino acids and sequences thereof in the receptor molecules that correspond to these domains and contribute to the activities identified for the domains. Thus, the previous identification of steroid receptor trans-activation regions resulted only from a demonstrated loss of activity via deletion or insertional mutagenesis, but in no case have the properties of the regions themselves been confirmed in assays that reflect a dominant gain of function. See also Ptashne, Nature 335, 683 (1988).
Thus, Godowski et al., Science 241, 812 (1988), report results that show that the glucocorticoid receptor contains at least one "enhancement domain" other than that overlapping the segment of DNA to which the receptor binds (i.e., the glucocorticoid response element or "GRE") and that this second domain occupies a region near the receptor amino-terminus. Similarly, Webster et al., Cell 54, 199 (1988) report on an inducible transcription activation function of the estrogen and glucocorticoid receptors, and these researchers speculate that the relative positions of the ligand-binding and DNA-binding domains are, not important for the trans-activation by the receptor. Yet, these researchers admit that they have no definition of the exact location and nature of what they call the "hormone-inducible activating domain" responsible for this inducible transcription activation function and they provide no characterization of the domain and no description of how, in molecular terms, it might contribute to trans-activation.
As a starting point for the present invention, Giguere et al., supra, demonstrated loss of activity in mutants of the glucocorticoid receptor, provided by DNAs resulting from random site-mutagenesis at several locations in a cDNA encoding the receptor. The loss of activity observed by Giguere et al. in this study was in an assay measuring transcription activity from a promoter operatively associated with a GRE so that transcription from the promoter was affected by glucocorticoid hormone receptor binding to the GRE. As a follow-up, Hollenberg et al., supra, deleted regions in the receptor molecule, again demonstrating overall loss of transcription activity induced by such removal of stretches of amino acids.
In PCT International Application No. US89/05419, filed Nov. 29, 1989, and corresponding to U.S. patent application Ser. No. 278,614, filed Nov. 30, 1988, as well as Hollenberg and Evans, supra, domain(s) responsible for trans-activation ("trans-activation" domains, i.e., domains responsible for the trans-acting transcription-activating activity of an hormone receptor at a promoter whose transcription activity is affected by the receptor) was (were) identified and characterized, and the characterization of such domain(s) in respect of amino acid composition and sequence was developed, to explore the functional interaction of the domain(s) with both the DNA-binding and ligand-binding domains of a given receptor, and finally, to exploit such knowledge to provide receptor analogs with increased trans-activating activity in comparison with that of the corresponding, naturally occurring receptor.
The present invention makes further use of the information provided by the invention disclosed in PCT International Application No. US89/05419 and Hollenberg and Evans, supra.
The human glucocorticoid receptor (hGR) has served as a prototype, model receptor for studying regulation of gene transcription by receptors of the steroid/thyroid hormone superfamily. The DNA-binding and ligand-binding functional domains of the hGR were first defined and the corresponding domains of other members of the superfamily identified in part on the basis of the results with the hGR. Further, it has been found that these DNA-binding and ligand-binding domains are modular in that, for example, the ligand-binding domain of a first hormone receptor (e.g., hGR) may be swapped with a ligand-binding domain of a second hormone receptor (e.g., hTR (human thyroid hormone receptor)) to produce a hybrid receptor the DNA-binding domain of which retains specificity for its cognate response element (GRE) in DNA, but which trans-activates only in the presence of the hormone (thyroid hormone, in this case) specific for the second receptor. Thus, reference herein to the "naturally occurring" receptor corresponding to a receptor analog of the invention means the naturally occurring receptor with the DNA-binding domain that is closest in primary sequence to the DNA-binding domain of the analog.
While trans-activation by the hGR (and other receptors of the steroid/thyroid hormone receptor superfamily) has been examined and elucidated in considerable detail, relatively little is known about "trans-repression" by the hGR or other receptors of the superfamily (i.e., trans-acting repression of transcription from a promoter, transcription from which is subject to repression by such a receptor).
Among their many effects in development and the reproductive, hepatic, metabolic, nervous and other systems, glucocorticoid hormones help determine neural crest cell fate in the developing sympathoadrenal system in part by repressing the induction of neural-specific genes (See Stein et al., Dev Bio 127, 316 (1988) and Anderson et al, Cell 47, 1079 (1986).) Also glucocorticoid hormones modulate the hypothalamic-pituitary-adrenal axis by inhibiting second messenger-induced peptide hormone induction. Recently, Akerblom et al. (Science 241, 350 (1988)) showed that the hGR negatively regulates (i.e., represses transcription from) the cAMP-inducible alpha glycoprotein hormone promoter in asteroid- and DNA-binding dependent manner. Wild-type expression is initiated by transcription from a promoter of Just 168 base pairs (termed alpha168). Basal expression of alpha glycoprotein hormone in placental cells is mediated by factors bound to a 36 base pair palindromic cyclic AMP response element (CRE) cooperating with proteins binding to a 25 base pair tissue-specific element (TSE). Expression may be further enhanced through the CRE by the elevation of intracellular cyclic AMP levels. The hGR represses both the basal and cAMP enhanced transcription in a glucocorticoid-dependent fashion. The transacting elements to which the hGR binds have been defined and are related to the consensus GRE sequence that has been defined in studies of trans-activation by the hGR. Similar research is reported by Sakai, et al., Genes and Development 2, 1144 (1988).
It is an object of the present invention to provide novel hormone or hormone-like receptor analogs that have trans-acting, transcription-repressing activity, at promoters whose transcription activity is capable of being repressed by the corresponding, native (i.e., naturally occurring) receptor. The novel receptor analogs are characterized in that they possess domains at their C-termini that differ in certain ways from the corresponding domains in the corresponding, native receptors.
It is a further object of the invention to provide novel, model, cellular and transgenic-animal systems of diseases, including human diseases, associated with the inability to respond properly to the hormone, which is the natural ligand of a naturally occurring receptor. It is still a further object of the invention to use these model systems to screen for compositions that are effective in treating such diseases. These cellular and transgenic-animal, model systems employ certain receptor analogs which have been found to have certain trans-repression-related activities, particularly transcription-repression activity greater than that of the corresponding, naturally occurring receptor, and DNAs of the invention, from which such analogs can be expressed in mammalian cells. Novel cells of the invention, including cells in novel transgenic animals of the invention which comprise novel cells of the invention, in which such receptor analogs are expressed will respond aberrantly to the hormone corresponding to the receptor analog because trans-repressing activities by the receptors (including the analog)for the hormone in the cells will aberrantly dominate trans-activating activities by such receptors.
It is a further object of the invention to use receptor analogs, which have been identified to have certain trans-repression activities, and DNAs of the invention, from which such analogs can be expressed in mammalian cells, in gene therapy to treat diseases associated with the inability to respond properly to the hormone, which is the natural ligand of the naturally occurring receptor corresponding to the analog. The effect of having the receptor analog in a subset of the cells of a mammal (including a human) treated by such gene therapy, would be to modulate the response of the mammal to the hormone to a more nearly normal response.
The invention has as still another object novel, functional assay systems, which employ the receptor analogs of the invention, whose transcription-repression activity is ligand (e.g., hormone-)dependent, novel DNAs of the invention, from which such analogs can be expressed, and novel cells of the invention, in which such DNAs are expressed, to screen for ligands that are effective in inducing the activities of the naturally occurring receptor corresponding to the analogs. Such functional assay systems to screen for such ligands are described in PCT International Publication No. WO 88/03168 and European Patent Application Publication No. 0 325 849.